Membrane fractions active in poliovirus RNA replication contain VPg precursor polypeptides

The poliovirus specific polypeptide P3-9 is of special interest for studies of viral RNA replication because it contains a hydrophobic region and, separated by only seven amino acids from that region, the amino acid sequence of the genome-linked protein VPg. Membraneous complexes of poliovirus-infected HeLa cells that contain poliovirus RNA replicating proteins have been analyzed for the presence of P3-9 by immunoprecipitation. Incubation of a membrane fraction rich in P3-9 with proteinase leaves the C-terminal 69 amino acids of P3-9 intact, an observation suggesting that this portion is protected by its association with the cellular membrane. These studies have also revealed two hitherto undescribed viral polypeptides consisting of amino acid sequences of the P2 and P3 regions of the polyprotein. Sequence analysis of stepwise Edman degradation show that these proteins are 3b/9 (Mr 77,000) and X/9 (Mr 50,000). 3b/9 and X/9 are membrane bound and are turned over rapidly and may be direct precursors to proteins P2-X and P3-9 of the RNA replication complex. P2-X, a polypeptide void of hydrophobic amino acid sequences but also found associated with membranes, is rapidly degraded when the membraneous complex is treated with trypsin. It is speculated that P2-X is associated with membranes by its affinity to the N-terminus of P3-9.     

Construction and properties of intertypic poliovirus recombinants: first approximation mapping of the major determinants of neurovirulence

An attempt was made to map, in a general way, the region of the poliovirus genome that is responsible for the neurovirulent and attenuated phenotypes of different virus strains. A set of four recombinants was investigated, one described previously (E. A. Tolskaya, L. I. Romanova, M. S. Kolesnikova, and V. I. Agol, 1983, Virology 124, 121-132) and three obtained in the present work with the following genetic structure: a 5' end-adjacent segment of the genome derived from either a virulent strain (452/62 3D), or from an attenuated strain (Leon-2) of poliovirus type 3, the remaining RNA sequences being derived from either a virulent strain (Mgr), or an attenuated strain (LSc-gr3) of poliovirus type 1. The crossover points in the recombinant genomes were centrally located, somewhere between the gene(s) that determines antigenic specificity of the virus and the locus that determines resistance of virus multiplication to low doses of guanidine. The recombinant nature of the newly selected clones was definitively established by mapping RNase T1 oligonucleotides of their genome. The recombinants were characterized with respect to their ability to produce infectious progeny and synthesize viral RNA at an elevated temperature. Neurovirulence of the recombinants was assayed by intracerebral inoculation of monkeys. Irrespective of the origin of the 3' end-adjacent segment of the genome, the recombinants that inherited the 5' end-adjacent segment from the neurovirulent parent were neurovirulent, whereas the recombinants with the 5' end-adjacent segment derived from the attenuated parent were not. The results suggest that the major determinants of neurovirulence of these recombinants (and by inference, of their parental viruses) reside in the 5' end-adjacent segment of poliovirus genome, known to code for capsid proteins.     

Cloning of bovine rotavirus (RF strain): nucleotide sequence of the gene coding for the major capsid protein

The genes of the RF strain of bovine rotavirus have been cloned into pBR 322 following the synthesis and hybridization of cDNA transcribed from both strands of in vitro polyadenylated genomic RNA. Cloned rotavirus DNAs were assigned to most of the 11 genomic RNA segments by Northern blot hybridization. The complete sequence of gene 6 that codes for the major inner capsid protein has been determined. The gene is 1356 nucleotides long and possesses an unique long open reading frame that could encode a protein (397 amino acids) of similar size to the known gene 6 product. Comparison of the RF bovine rotavirus gene 6 sequence with the sequence of the simian rotavirus gene 6, showed these genes to be very similar in nucleotide sequence (87% homology). Most of the base changes are silent and the predicted amino acid sequences are almost identical (97% homology).     

Studies on the in vitro uncoating of poliovirus. II. Characteristics of the membrane-modified particle.

Interaction of type I poliovirus with an extract of isolated HeLa cell membranes resulted in a modification of the viral capsid. As a consequence of the modification, the capsid became sensitive to proteases and detergents, and the sedimentation value of the virion was slightly diminished. Treatment of the modified particle with chymotrypsin reduced its sedimentation value and, although the RNA genome was still encapsidated, it was degradable by RNase. Treatment of the membrane-modified particle with detergent (sodium dodecyl sulfate, SDS) disengaged the viral RNA as an intact (i.e., 35 S) molecule. Each of the three serotypes of poliovirus was modified when treated with membrane extract. They differed, however, in the effect of secondary treatment with chymotrypsin, viz., type I was further modified, but types II and III underwent degradation. When secondary treatment was with SDS, all three released 35 S RNA. The above in vitro reactions of the membrane-modified particle are discussed as possible counterparts of the in vivo uncoating phenomenon.     

Structural studies of the RNA component of the poliovirus replication complex. I. Purification and biochemical characterization.

Substantial purification of the RNA component of the poliovirus replication complex is achieved by agarose chromatography of the high salt precipitated replication complex. The viral RNA isolated in the agarose-purified fraction is predominantly derived from in vivo replicating structures. Purified poliovirus RNA polymerase activity is associated with an endogenous RNA template that resembles poliovirus replicative intermediate RNA.     

Natural variants of the Sabin type 1 vaccine strain of poliovirus and correlation with a poliovirus neutralization site

Independent substitution mutations have been detected in capsid polypeptide VP1 of the type 1 oral poliovirus vaccine isolated from normal infant vaccine recipients. These mutations map at amino acid residues 142 and 147 of VP1, a region only minimally hydrophilic. A synthetic peptide, corresponding to residues 141 to 147 of VP1 was synthesized, conjugated to a carrier polypeptide of bovine serum albumin. The conjugate was found to elicit a weak poliovirus neutralizing antibody response. It was also capable of priming the immune system for the production of IgG-type antibodies able to neutralize greater than 99.999% of infectious type 1 virus. It is suggested that region 141 to 147 of VP1 may be involved in neutralization of the virus and that the mutants may have accumulated by antibody selection.     

Structural studies of the RNA component of the poliovirus replication complex. II. Characterization by electron microscopy and autoradiography.

The endogenous RNA component of the purified poliovirus replication complex was characterized in the electron microscope after cytochrome spreading. This RNA species has a double-stranded RNA core equal in length to poliovirus replicative form RNA, with 0 to 6 single-stranded tails per molecule. DNase I, RNase, and base treatment confirmed the double-stranded RNA nature of these molecules, which are not observed in extracts from uninfected or infected, guanidine-inhibited cells. Electron microscope autoradiography verified that these double-stranded RNA structures are the site of in vitro and in vivo viral RNA synthesis. After RNA synthesis in vitro, the double-stranded core is unchanged, but the number of tails decreases and the branch points are localized towards the ends of molecules. These results demonstrate that the RNA component of the active purified replication complex is analogous to replicative intermediate RNA and favor the double-stranded model for the in vivo poliovirus replication complex.     

Biochemical analysis of the capsid protein gene and capsid protein of tobacco etch virus: N-terminal amino acids are located on the virion's surface

The sequence of the 1491 nucleotides found at the 3' end of the genome of the highly aphid-transmissible (HAT) isolate of tobacco etch virus (TEV) has been determined. The nucleotide sequence of the capsid protein gene has been identified and compared with the corresponding region of the not-aphid-transmissible (NAT) isolate of TEV and with pepper mottle virus (PeMV). The deduced amino acid sequences of the two TEV capsid proteins displayed 98% homology and a 66% homology with PeMV capsid protein. Three of the six amino acid differences between the capsid proteins of the two TEV isolates occurred near the N terminus of the protein. Biochemical and immunological evidence suggested the N-terminal 29 amino acids of the capsid protein were hydrophilic and were located at or near the virion's surface.     

Immunological and physicochemical studies of influenza matrix (M) polypeptides.

Immunologically active preparations of M polypeptides were recovered from influenza A viruses and had a sedimentation coefficient of 3.3 s. The type-specific antigenic determinants of the M polypeptide were resistant to denaturation by heat at 100° for 2 min in the presence of detergents and to treatment with acidic-chloroform methanol. Mild proteolysis with trypsin or caseinase C produced fragments of 13,000 and 6,000 daltons. The 13,000-dalton fragments possessed some identical antigenic determinants to the whole 25,000-dalton M molecule and partially absorbed antibody to the 25,000-dalton polypeptide from a specific antiserum. Immunization of ferrets with M polypeptide or M polypeptide fragments had no preventative or enhancing effect on influenza virus infection, whereas immunization with homologous bromelain-released HA polypeptides conferred absolute protection to virus challenge.     

Studies on the in vitro uncoating of poliovirus. III. Roles of membrane-modifying and -stabilizing factors in the generation of subviral particles

A quantitative assay for defective-interfering (DI) particles of Sindbis virus (SV) was developed and shown to have an efficiency comparable to that of the plaque assay for infective virus. The assay was used to quantitate the kinetics of ultraviolet inactivation of the interfering activity of DI particles. For SV_BP19 (a virus stock produced by 19 serial undiluted passages in BHK cells) and SV_A17 (produced by 7 serial undiluted passages in A. albopictus cells) the interfering activity was seven- and twofold, respectively, more resistant to inactivation by uv light than was infectivity. For the DI particles in SV_BP19 the uv target size correlated very well with the size of the DI genome RNA (MW = 0.59 and 0.63 × 10⁶) but for the DI particles in SV_AP7 the uv target size was 25–30% smaller than the DI genome (MW = 3.2 × 10⁶). These results suggest that the entire genome of the DI particle in SV_AP7 may not be required for interference. In contrast to the results obtained with DI particles, the interfering capacity of a nondefective ts mutant was almost as sensitive to inactivation as was infectivity.     

Studies on the in vitro uncoating of poliovirus : I. Characterization of the modifying factor and the modifying reaction

A method is described for obtaining a membrane-rich fraction from HeLa cells that can induce a modification in poliovirus characterized by (a) the loss of infectivity and (b) the development of sensitivity of the viral RNA to ribonuclease after treatment with chymotrypsin.Physico-chemical characterization of the modifying factor indicates that it is, or is bound to, a high molecular weight component of the plasma membrane. Modifying activity is lost after exposure to ether, deoxycholate, low pH, trypsin, or brief sonication. Treatment of intact cells with trypsin prior to fractionation, yields a final product devoid of activity. Activity is less readily lost after treatment with alkali. About 50% of the activity is lost after several cycles of freeze-thaw, without further reduction on continued treatment. At 5°, activity gradually diminishes to an undetectable level in about 1 week. At higher temperatures, e.g., 25–45°, activity is lost more rapidly. Neither DNAse nor RNAse has any effect on activity.The modifying reaction proceeds optimally at pH 8. The reaction is readily inhibited by nonionic and ionic compounds. Sucrose and glycerol at a concentration of 5% inhibit almost completely. Ionic compounds at a molarity of 0.05 are strongly inhibitory. On a molar basis, divalent cations are 10-fold more inhibitory than monovalent cations. Kinetic studies indicate that at 25° there is no measurable activity, but between 30 and 37° the reaction has an activation energy of 60 kcal/mole.The modifying factor is active on a wild-type and an attenuated type I poliovirus, but inactive on types II and III viruses.Evidence has been obtained that the membrane extract contains two factors, one with modifying activity, and another that can competitively inhibit the modification reaction.     

Characterization and localization of CIV polypeptides

In order to detect the structural proteins linked with disulfide bonds, CIV was solubilized and electrophoresed under nonreducing conditions in the first dimension and then under reducing conditions in the second dimension. The viral polypeptides linked originally with disulfide bonds were separated into subunits. The complexes were trimers (P'50) or dimers (P60 and P10). The apparent molecular weights of P81, P53, and P49 changed significantly according to the composition of the lysis buffer used, suggesting that the differences in their molecular weights were due to conformational changes produced by reduction of their intramolecular disulfide bonds. Sulfhydryl-containing polypeptides (P'50-P50, P60, P100, and P33) were detected by N-[14C]ethylmaleimide, and the accessibility of these residues was analyzed after successive stripping of the CIV particle. Radioiodination of external polypeptides by [125I]iodosulfanilic acid shows only one intensively labeled spot corresponding to the P50 polypeptide, whereas P'50 was only slightly labeled. Six viral polypeptides P81, P60, P31, P17, P13, and P10 were revealed to possess high affinity for CIV DNA. A structural model of CIV is proposed and discussed.     

Intracellular distribution of poliovirus proteins and the induction of virus-specific cytoplasmic structures

In a susceptible cell, enteroviruses induce a vesiculated region (the "virus-induced vesicles") which is both the site of viral RNA synthesis as well as the site referred to morphologically, as the "cytopathic effect." Proteins of poliovirus (type I, Mahoney) were shown to migrate into the region of the virus-induced vesicles of infected HEp-2 cells. Five proteins (P2-5b, P3-4b, P3-6a, P3-7c, P3-9) were found to be associated with the vesicles themselves, either as intrinsic membrane protein (P3-9) or in a soluble form within the vesicles (P3-4b, P3-7c, and, partially, P3-6a) or bound to a DOC-resistant structure (P2-5b and a small amount of P3-6a). Partial inhibition of the cleavage of the viral polyprotein with ZnCl2 was used to alter the viral protein pattern within the cells. The data obtained indicate that P2-5b is the protein responsible for the formation of the virus-induced vesicles.     

Isolation and characterization of defective-interfering particles of poliovirus Sabin 1 strain

Defective-interfering (DI) particles of the Sabin strain of type 1 poliovirus were generated on serial high m.o.i. passaging. The deletions, measured by agarose gel electrophoresis, appeared to comprise approximately 10% of the total genome. Analysis of the RNAs, after digestion with RNase T1, by two-dimensional polyacrylamide gel electrophoresis revealed that the locations of the deleted genome regions were similar to those of the DI particles of the Mahoney strain of type 1 poliovirus (A. Nomoto, A. Jacobson, Y. F. Lee, J. Dunn, and E. Wimmer, (1979), J. Mol. Biol. 128, 179-196). Taking the known nucleotide sequences of the total genome and large RNase T1-resistant oligonucleotides into account, the deletions of almost all DI RNAs were found to exist between nucleotide positions 1307 and 2630, a genome region encoding capsid polypeptides VP2, VP3, and VP1. In cells coinfected with the purified DI particles and the Sabin strain of type 2 or type 3 poliovirus, particles containing the DI genomes were effectively produced. These results suggest that encapsidation signals are conserved in all three serotypes of polioviruses. However, only a very small amount of similar DI particles appeared to be produced in cells coinfected with coxsackie virus B1, although the genomes of polioviruses and coxsackie viruses have common sequences and therefore these viruses are considered to have arisen from a common ancestor. These data may suggest differences in encapsidation signals between polioviruses and coxsackie viruses.     

Topographic analysis of tobacco etch virus capsid protein epitopes

Monoclonal antibodies have been prepared, which react with capsid protein of an aphid-transmitted isolate of tobacco etch virus (TEV). Ten different monoclonal antibodies were characterized with reference to (1) antibody class, (2) reactivity with different plant virus antigens, (3) the spatial relationship between epitopes, and (4) whether these epitopes were located on the exterior surface of the virion. Three monoclonal antibodies were specific for TEV isolates. These monoclonal antibodies reacted with epitopes exposed on the external surface of the TEV particle. Seven monoclonal antibodies reacted with a variety of different potyviruses including TEV, potato virus Y, tobacco vein mottling virus, pepper mottle virus, watermelon mosaic virus II, and maize dwarf mosaic virus. In general, these seven monoclonal antibodies defined epitopes not readily accessible on the virion surface.     

Antigenic conservation and divergence between the viral-specific proteins of poliovirus type 1 and various picornaviruses

Immuneprecipitation analyses of various picornavirus-infected cell lysates were performed using antisera to poliovirus type 1-specific structural and nonstructural proteins. The results showed differing patterns of antigenic conservation and divergence. However, the VP3 and 2C polypeptides were strongly antigenically conserved among the large majority of these viruses. This conservation was especially notable given the degree of divergence exhibited by the other viral proteins and may be due to environmental pressure exerted by interaction with the host cell. The results, furthermore, allowed for an analysis of the evolutionary relationship of the tested viruses. This analysis showed a particularly strong antigenic relationship between the proteins of the poliovirus group and coxsackievirus A21 as well as a weaker, but significant, relationship with coxsackieviruses B1 and B3.     

Human cytomegalovirus polypeptides stimulate neutralizing antibody in vivo

At least three human cytomegalovirus polypeptides are targets for virus neutralizing antibody; a single protein of 86,000 molecular weight (p86) and two coimmunoprecipitating proteins of 130,000 and 55,000 molecular weight (p130/55). These polypeptides have been isolated by immunoaffinity chromatography and tested for immunogenicity in guinea pigs. Neutralizing antibody was detected after immunization with both p86 and p130/55. Hyperimmune sera to p130/55, but not p86, were dependent upon guinea pig complement for virus neutralization.     

Neutralization of poliovirus by a monoclonal antibody: kinetics and stoichiometry

First-order kinetics of neutralization have usually been interpreted as evidence that a single antibody, binding at a critical site, neutralizes the infectivity of a virus particle. In such a case, if all the binding sites were critical, an average of one antibody bound per virion would be required to reduce the infectivity of a virus sample to 37% (1/e) of its initial infectivity. However, in the work reported here using a monoclonal antibody to poliovirus which inactivated with first-order kinetics, an average of four bound antibodies were required. These results are consistent with two different models: one in which only one-fourth of the antibody binding sites on the virion are critical for neutralization; the other, in which none of the sites is critical, but neutralization takes place instead in a stepwise fashion in which each bound antibody reduces the infectivity by a factor of 3/4. The maximum binding capacity of the virion for this monoclonal antibody was approximately 30 molecules. Since the 60 protein subunits of the poliovirus capsid are related by 30 twofold axes of symmetry, it is proposed that each monoclonal antibody binds bivalently to two protein subunits related by a twofold axis. Such a binding mode would crosslink pentamers, the basic structures in picornaviral assembly and dissociation. It is proposed that pentamer crosslinking is an important element in neutralization by this monoclonal antibody. Another antibody, which may neutralize by a different mechanism, is also discussed briefly.     

Biosynthesis of reovirus-specified polypeptides: the reovirus s1 mRNA encodes two primary translation products

Reovirus serotypes 1 (Lang strain) and 3 (Dearing strain) code for a hitherto unrecognized low-molecular-weight polypeptide of Mr approximately 12,000. This polypeptide (p12) was synthesized in vitro in L-cell-free protein synthesizing systems programmed with either reovirus serotype 1 mRNA, reovirus serotype 3 mRNA, or with denatured reovirus genome double-stranded RNA, and in vivo in L-cell cultures infected with either reovirus serotype. The synthesis of p12 in vivo was insensitive to actinomycin D, and occurred at similar times after infection as the previously identified reovirus encoded lambda, mu, and sigma polypeptides. Pulse-chase experiments in vivo, and the relative kinetics of synthesis of p12 in vitro, indicate that it is a primary translation product. Fractionation of reovirus mRNAs by velocity sedimentation and translation of separated mRNAs in vitro suggests that p12 is coded for by the s1 mRNA, which also codes for the previously recognized sigma 1 polypeptide. Synthesis of both p12 and sigma 1 in vitro in L-cell-free protein synthesizing systems programmed with denatured reovirus genome double-stranded RNA also suggests that these two polypeptides can be coded by the same mRNA species. The Mr approximately 12,000 polypeptide was not a detectable structural component of purified virions, and antiserum prepared against purified reovirions did not immunoprecipitate p12. It is proposed that the Mr approximately 12,000 polypeptide encoded by the S1 genome segment be designated sigma 1bNS, and that the polypeptide previously designated sigma 1 be renamed sigma 1a.